Project Summary/Abstract Triple-negative breast cancers (TNBCs) account for 15 to 20% of breast cancer cases, are often clinically aggressive, and typically exhibit high rates of recurrence and mortality. Approximately one third of TNBC cases feature genetic inactivation of the PTEN tumor suppressor, and a considerably larger percentage exhibit PTEN deficiency due to epigenetic and post-transcriptional mechanisms. Since PTEN is the major negative regulator of PI3K activity in cells, PTEN-deficient cancers are characterized by hyper-activated PI3K signaling and were therefore expected to respond to therapeutic PI3K inhibition. However, pan-PI3K inhibitors have demonstrated poor activity against PTEN-deficient cancers in clinical studies. Notably, we have previously shown that tumors driven by loss of PTEN are uniquely dependent on the p110? isoform of PI3K. This finding likely explains why PI3K inhibitors first tested on PTEN null tumors failed in the clinic, as they primarily targeted p110? and did not adequately inhibit p110?. New p110?-specific compounds, on the other hand, are now showing clinical promise in PTEN-deficient solid tumors. In attempting to understand the molecular mechanisms that uniquely couple PTEN loss to p110? activation, we have uncovered a set of molecular mechanisms that not only explain how p110? is activated in response to loss of PTEN, but also suggest potential mechanisms of resistance and drug targets that could be targeted in combination with p110? inhibition. In this project, we will rigorously evaluate therapeutic strategies combining p110? inhibitors and targeted therapies to the targets we have identified using multiple genetically-engineered mouse models (GEMMs) of TNBC with PTEN deficiency, as well as in a panel of patient-derived xenograft (PDX) models of TNBC and ex vivo primary organoid cultures. Our preliminary data and pilot in vivo studies have provided encouraging results indicating that targeting these pathways in combination with p110? inhibition results in better response. Moreover, we found that p110? is crucial for mediating immune evasion in PTEN-deficient TNBC and that p110? inhibition strongly synergizes with anti-PD-1 immune checkpoint blockade to inhibit tumor growth. These findings revealed a previously unexplored role for p110? signaling in immune modulation, and provide a strong rationale for this combination treatment in PTEN-deficient TNBC. We will therefore investigate combined p110? inhibition and immunotherapy in GEMMs and organoid models of PTEN-null TNBC. Furthermore, we will research the molecular mechanisms by which p110? mediates tumorigenesis and immune evasion. As we move forward, we expect that the results from this project will provide important pre-clinical information for the design of future clinical trials of these combination therapies.